Our preliminary results have shown a direct suppression of IL-17 by 1,25(OH)2D3 as well as reversal of paralysis and inhibition of progression of EAE [a murine model of multiple sclerosis (MS)] by 1,25(OH)2D3 which is associated with an inhibition of IL-17. We propose to examine the mechanisms involved. We hypothesize that understanding the mechanisms involved will result in new concepts in our understanding of the interaction between the vitamin D endocrine system and the immune system that may suggest therapeutic targets for the control of MS and other TH17 dependent inflammatory diseases including inflammation induced bone loss. In Specific Aim 1 we will examine the effect of in vivo treatment of EAE mice with 1,25(OH)2D3 on the production of IL-17 and other cytokines (brains and spinal cords as well as splenocytes and lymph nodes will be isolated from EAE mice and the effect of 1,25(OH)2D3 on the production of various cytokines including those produced by TH17 cells (IL-17A, IL-17F, IL-21, IL-22), TH2 cells (IL-4, IL-5), regulatory T cells (IL-10 and TGF?) and innate immune cells (IL-23, IL-12, IL6 and the anti-inflammatory cytokine, IL-27) will be assessed. In preliminary results we noted for the first time that 1,25(OH)2D3 has a direct inhibitory effect on activated IL-17 expression and transcription. The mechanisms involved will be examined (1,25(OH)2D3 may mediate this repression by inhibiting activation mediated by NFAT, Runx1 and ROR gamma transcription factors). Genome-wide analysis of NFAT, Runx1 and vitamin D receptor (VDR) binding sites in CD4+T cell DNA isolated from EAE mice treated with vehicle or 1,25(OH)2D3 using ChIP-seq will also be done. These studies will enable us to identify new target genes and to assess how functional relationship among genes involved in immune function may be altered after 1,25(OH)2D3 treatment. These studies would provide mechanisms for the reversal of paralysis by 1,25(OH)2D3. It is possible that the mechanisms we identify may reflect more general mechanisms involved in a therapeutic role of 1,25(OH)2D3 in the control of pathological immune responses. In Specific Aim 2, since clinical studies are being done treating MS patients with high dose vitamin D, we also propose to examine the effect of high dose dietary vitamin D on paralysis, on the progression of EAE and the production of IL-17 and other cytokines. These studies provide a unique opportunity to combine the expertise of the Steinman lab in multiple sclerosis and immunology and the Christakos lab in vitamin D to increase our understanding of the interaction between the vitamin D endocrine system and the immune system. Findings from these studies may suggest new therapeutic targets and treatment strategies for MS and other TH17 dependent inflammatory diseases. This application is appropriate for the R21 mechanism which supports projects that involve considerable risk but may lead to a breakthrough in a particular area that could have a major impact on a field of biomedical or clinical research.